X-ray crystallographic investigation of some biologically important molecules

Abstract

The thesis entitled “X-ray Crystallographic Investigation of Some Biologically Important Molecules” consists of two parts. Part I, with four chapters, deals with the structure and conformation of a dipeptide, glycyl-DL-phenylalanine (Chapter 1); 4-azatricyclo[4.4.0.05,8]decan-5-one containing a 3-peptide unit (Chapter 2); and an amino acid, L-serine (Chapter 4). Chapter 3 surveys accurately solved structures to find out the possible effect of hydrogen bonding on the geometry of the peptide unit.

Part I X-ray crystallographic investigations of simple peptides—linear and cyclic—are important, since such studies provide valuable information regarding the geometry of the peptide fragment, the mode of packing of the side chains, and the effect of intermolecular interactions such as hydrogen bonding on the conformation of the molecule.

The crystals of glycyl-DL-phenylalanine are orthorhombic, a = 9.241(2) Å, b = 28.41(1) Å, c = 8.602(2) Å; space group Pbca, Z = 8. The structure has been solved using direct methods with diffractometer data and has been refined using full matrix least-squares to an R-value of 0.041 for 2271 reflections. The results are discussed in Chapter 1.

The dimensions of the peptide group are in good agreement with the average values in other peptides reported by Marsh and Donohue, the largest difference being in the C=O bond, which is 0.02 Å shorter. The possible reasons for the shortening are discussed. The peptide group is appreciably non-planar, with the torsion angle about the peptide bond being -170.2(2)° for the L-molecule. The phenylalanine side chain goes over to position III with the torsion angle = -59.7°, in accordance with the predictions of semi-empirical energy calculations. The organization of the molecules is such that the hydrogen bonds between molecules of the same configuration are stronger than those between molecules of opposite configuration.

The crystal and molecular structure of 4-azatricyclo[4.4.0.05,8]decan-5-one was analyzed with a view to obtain accurate information of the geometry of the cis-peptide unit and to compare the geometry under different conditions. The crystals are monoclinic, a = 6.662(6) Å, b = 13.36(2) Å, c = 8.606(9) Å, ? = 98.97(2)°; space group P21/n, Z = 4. The structure has been solved using direct methods with diffractometer data and has been refined by block-diagonal least-squares to an R-value of 0.035 for 1066 reflections. The results are presented in Chapter 2.

The bond angles C(3)-N(4)-C(5) and C(6)-C(5)-N(4) are found to be significantly smaller than the corresponding angles for the standard cis-peptide unit. Similar decreases in angles are also found when the cis-peptide unit forms a part of four- and five-membered rings. The cis-peptide unit is significantly non-planar, with the torsion angle about the peptide bond being 14.5(3)°. The C(sp3)-C(sp3) single bonds linking carbon atoms to which three heavy atoms are attached are found to be significantly longer than the standard C–C single bond. The bicyclo-octane ring system present in the molecule has an approximate D3(32) symmetry, and its conformation has been compared with that found in other structures.

The potential energy required for non-planar distortions of the peptide unit has been evaluated by two groups of workers. The Zurich group of Dunitz and co-workers has evaluated the potential based on structural and spectroscopic evidence. The Bangalore group of Ramachandran and co-workers has obtained the potential energy using the INDO approximation. The results of these two approaches are compared in Chapter 3, and the agreement between them is found to be remarkably good. Both approaches predict a planar equilibrium configuration for the peptide unit, and small non-planar distortions at the peptide nitrogen atom can be introduced at a modest energy cost.

A statistical analysis of accurately solved crystal structures has been made, and it reveals that the deviation of the hydrogen atom of the peptide unit from the plane defined by the atoms C, N, and O is such that a more linear hydrogen bond involving the peptide hydrogen is achieved. The position of the hydrogen atom as determined experimentally is found to correlate better with the position of the acceptor atom than with the theoretically expected hydrogen position, corresponding to the minimum energy for a given torsional rotation about the peptide bond.

Chapter 4 deals with the crystal and molecular structure of L-serine. The crystals are orthorhombic, a = 8.58(1) Å, b = 9.34(1) Å, c = 5.61(1) Å; space group, Z = 4. The structure has been solved using the Patterson search method. The search fragment consisted of the planar carboxyl group together with the nitrogen atom, and its orientation in the unit cell was determined using Nordman’s vector space search. The correct location of the fragment with respect to the 2-fold axes was determined using the Q-functions. The structure has been refined using block-diagonal least-squares to an R-value of 0.106 for 375 visually estimated reflections. A comparison of the hydrogen bonding scheme in L-serine, DL-serine, and L-serine monohydrate reveals that hydrogen bonding affects the dimensions of the carboxylate ion, i.e., stronger hydrogen bonding is associated with the longer O–O bond. The conformation of the molecule is compared with the predictions of semi-empirical energy calculations.

Part II During the course of synthesis of rigid or semi-rigid analogs of acetylcholine, 1-diphenylmethylazetidin-3-ol was obtained by Ohatterjee and Triggle. The compound is a good anti-convulsant agent. The crystals are triclinic, a = 8.479(2) Å, b = 17.294(4) Å, c = 10.606(3) Å, ? = 118.59(2)°, ? = 100.50(2)°, ? = 89.63(2)°; space group P1, Z = 4, i.e., two independent molecules in the asymmetric unit. The E values were corrected for molecular scattering effects. The structure has been solved using direct methods and has been refined using full-matrix least-squares to an R-value of 0.044 for 2755 reflections. The results are discussed in Chapter 5.

The bond lengths, bond angles, and the angle of puckering of the azetidine rings obtained in the present study are compared with those in other structures containing the ring. It is found that the C–C bond lengths show a larger variation than the C–N bond lengths. The angle of puckering of the four-membered ring ranges from 180° to 153°. The conformational features of the azetidine ring are compared with those of the cyclobutane ring. The two independent molecules present in the asymmetric unit differ in the conformation of the phenyl rings with respect to the four-membered ring. Semi-empirical energy calculations have been carried out to explain this difference.

Coleonol is an active diterpenoid isolated by Tandon and Dhar from the plant Coleus barbatus. It has excellent hypotensive and spasmolytic activities. The structure of the compound could not be established unambiguously by spectroscopic methods. Hence, single-crystal X-ray diffraction study of this pharmacologically important compound was undertaken. The crystals are orthorhombic, a = 20.24(3) Å, b = 16.26(2) Å, c = 6.69(2) Å; space group P212121, Z = 4. The structure has been solved using direct methods. The E map revealed only the partial structure, and the rest of the atoms were located by successive difference electron density calculations sandwiched between least-squares refinement. The structure has been refined by block-diagonal least-squares to an R-value of 0.104 for 1400 visually measured reflections. The results are discussed in Chapter 6.

Atom C(15) is associated with two possible sites. The effect of axial substituents on the bond angles and torsion angles is discussed.

The program MULTAN (Germain, Main, and Woolfson) was used for solving the structures described in Chapters 2, 5, and 6. The program was written originally for a medium-sized computer with 52K words (128K bytes) memory. However, the author had access to a computer with only 16K words (64K bytes) memory and had to incorporate the MULTAN program into the small computer. The details are presented in Appendix A of the thesis. The Patterson search programs used to solve the structure reported in Chapter 4 are described in Appendix B. Appendix C contains an account of other programs developed by the author during the course of the investigation.

Based on the work carried out by the author, references to papers either already published or accepted for publication are given below:

The Crystal and Molecular Structure of Glycyl-DL-Phenylalanine. Marsh, R.E., Ramakumar, S., and Venkatesan, K., (1976). Acta Cryst. B32, 66.

The Crystal Structure of 4-azatricyclodecan-5-one. Venkatesan, K., Ramakumar, S., and Weber, H.P., (1975). Acta Cryst. A31, S103 (Supplement).

Determination of the Crystal and Molecular Structure of L-serine using Rotation and Translation Functions. Ramakumar, S., Venkatesan, E., and Shamala, U., (1973). Indian Journal of Pure and Applied Physics, 461.

The Crystal and Molecular Structure of 1-diphenylmethylazetidin-3-ol. Ramakumar, S., Venkatesan, E., and Rao, S.T., (1976). Acta Cryst. B (In Press).